Research article Dynamic rerouting of the carbohydrate flux is key to counteracting oxidative stress Markus Ralser*, Mirjam M Wamelink † , Axel Kowald* ¶ , Birgit Gerisch*, Gino Heeren § , Eduard A Struys † , Edda Klipp*, Cornelis Jakobs † , Michael Breitenbach § , Hans Lehrach* and Sylvia Krobitsch* Addresses: *Max Planck Institute for Molecular Genetics, Ihnestrasse 73, 14195 Berlin, Germany. † Department of Clinical Chemistry, Metabolic Unit, VU University Medical Center, Amsterdam, de Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. § Department of Cell Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria. ¶ Current address: Medical Proteome Center, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany. Correspondence: Markus Ralser. Email: ralser@molgen.mpg.de; Sylvia Krobitsch. Email: krobitsc@molgen.mpg.de Abstract Background: Eukaryotic cells have evolved various response mechanisms to counteract the deleterious consequences of oxidative stress. Among these processes, metabolic alterations seem to play an important role. Results: We recently discovered that yeast cells with reduced activity of the key glycolytic enzyme triosephosphate isomerase exhibit an increased resistance to the thiol-oxidizing reagent diamide. Here we show that this phenotype is conserved in Caenorhabditis elegans and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H) pool. Remarkably, another key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), is known to be inactivated in response to various oxidant treatments, and we show that this provokes a similar redirection of the metabolic flux. Conclusions: The naturally occurring inactivation of GAPDH functions as a metabolic switch for rerouting the carbohydrate flux to counteract oxidative stress. As a consequence, altering the homoeostasis of cytoplasmic metabolites is a fundamental mechanism for balancing the redox state of eukaryotic cells under stress conditions. BioMed Central Journal of Biology 2007, 6:10 Open Access Published: 21 December 2007 Journal of Biology 2007, 6:10 (doi:10.1186/jbiol61) The electronic version of this article is the complete one and can be found online at http://jbiol.com/content/6/4/10 Received: 21 May 2007 Revised: 7 August 2007 Accepted: 12 October 2007 © 2007 Ralser et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background Reactive oxygen species (ROS) cause damage to cellular processes in all living organisms and contribute to a number of human disorders such as cancer, cardiovascular diseases, stroke, and late-onset neurodegenerative disorders, and to the aging process itself. To cope with the fatal cellular consequences triggered by ROS, eukaryotic cells have evolved a number of defense and repair mechanisms, which are based on enzymatic as well as non-enzymatic processes and appear to be highly conserved from unicellular to multicellular eukaryotes. In bacteria and yeast, these anti- oxidant defense mechanisms are partially induced on the basis of changes in global gene expression [1,2]. However, a recent study analyzing a number of genetic and environ- mental perturbations in Escherichia coli demonstrated that the changes in the transcriptome and proteome are unex- pectedly small [3]. Moreover, the transcription of genes encoding enzymes capable of neutralizing ROS is not gener- ally increased in mammalian cells that are subjected to oxidative stress [4]. In all organisms studied, however, treatment with oxidants prompts immediate de novo post-translational modifica- tions of a number of proteins, probably affecting their local- ization and functionality. One of the key targets of those processes is the glycolytic enzyme glyceraldehyde-3-phos- phate dehydrogenase (GAPDH), which catalyzes the reversible oxidative phosphorylation of glyceraldehyde-3- phosphate (gly3p) to 1,3-bisphosphoglycerate. Remarkably, in response to various oxidant treatments this enzyme is inactivated and transported into the nucleus of the cell, and has been found S-nitrosylated, S-thiolated, S-glutathiony- lated, carbonylated and ADP-ribosylated in numerous cell types and organisms under these conditions [5-10]. Recently, we discovered that yeast cells with reduced cat- alytic activity of another key glycolytic enzyme, triose- phosphate isomerase (TPI), are highly resistant to the oxidant diamide [11]. This essential enzyme precedes GAPDH in glycolysis, catalyzing the interconversion of di- hydroxyacetone phosphate (dhap) and gly3p, the substrate of GAPDH, and a reduction in its activity results in an ele- vated cellular dhap concentration [12-14]. In this light, it is remarkable that the expression of a subset of glycolytic pro- teins and proteins implicated in related pathways is repres- sed, while the expression of a few enzymes involved in the pentose phosphate pathway (PPP), which is directly con- nected to the glycolytic pathway, is induced under oxidative stress conditions [1]. Furthermore, enhanced activity of the PPP has been observed in neonatal rat cardiomyocytes and in human epithelial cells under oxidative stress conditions [15,16]. Enzymes of the PPP are crucial for maintaining the cytoplasmic NADPH concentration, which provides the redox power for known antioxidant systems [17,18]. The observations above suggest that alterations in the carbohy- drate metabolism could be central for cellular protection against ROS and, moreover, that cells reroute the carbohy- drate flux from glycolysis to the PPP to counteract perturba- tions in the cytoplasmic redox state. However, direct evidence for this hypothesis is missing so far. By combining genetic and quantitative metabolite analyses along with in silico modeling, we present the first direct proof that eukary- otic cells indeed actively reroute the metabolic flux from glycolysis to the PPP as an immediate and protective response to counteract oxidative stress. Results Reduced intracellular TPI concentration results in enhanced oxidant resistance of Saccharomyces cerevisiae and Caenorhabditis elegans We reported earlier that a change of the amino acid isoleucine to valine at position 170 in the human TPI protein (TPI Ile170Val ) causes a reduction of about 70% in the enzyme’s catalytic activity [11]. Interestingly, we discovered that yeast cells expressing this human TPI variant exhibit increased resistance to the oxidant diamide (N,N,N’,N’- tetramethylazodicarboxamide, Chemical Abstracts Service (CAS) No. 10465-78-8) compared with isogenic yeast cells expressing wild-type human TPI, indicating that low TPI activity confers resistance to specific conditions of oxidative stress. The synthetic reagent diamide is known to oxidize cellular thiols, especially protein-integrated cysteines [19], provoking a rapid decrease in cellular glutathione and hence causing oxidative stress. To dissect the underlying mechanism, we first analyzed whether decreasing the expression level of wild-type human TPI would result in a similar phenotype. For this, we generated plasmids for the expression of wild-type human TPI under the control of established yeast promoters of different strengths, namely the CYC1, TEF1 and GPD1 promoters [20]. Subsequently, the ∆tpi1 strain MR100, which is deleted for the yeast TPI1 gene and is inviable on medium containing glucose as sole carbon source, was transformed with the respective plas- mids along with control plasmids encoding yeast TPI Ile170Val or yeast TPI. Single colonies were selected and the intracel- lular TPI concentration of plate-grown yeast cells was ana- lyzed (Figure 1a, left panel). As expected, yeast cells expressing the different TPI proteins under the strong GPD1 promoter had a higher TPI concentration compared with cells in which the expression was controlled by the interme- diate TEF1 or the weak CYC1 promoter. Next, we spotted the respective yeast cells onto medium supplemented with differing diamide concentrations. As shown in Figure 1a (right panel), yeast cells expressing human TPI under the control of the weakest promoter used, the CYC1 promoter, 10.2 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. http://jbiol.com/content/6/4/10 Journal of Biology 2007, 6:10 grew slowly on standard medium compared with the other yeast strains. Notably, growth of these cells on plates con- taining 1.6-1.8 mM diamide was comparable to the growth of control yeast cells expressing the TPI Ile170Val protein with reduced catalytic activity, demonstrating that a reduction in TPI expression or specific activity confers resistance against this oxidant. Furthermore, yeast cells expressing wild-type human TPI under the control of the intermediate TEF1 pro- moter grew on medium containing 1.8 mM diamide, albeit to a much lesser extent than yeast cells in which TPI expres- sion is controlled by the weak CYC1 promoter. This finding excludes the possibility that the observed oxidant resistance of yeast cells with CYC1-controlled TPI expression is based solely on their slower growth rate. In support of this, yeast cells in which the strong GPD1 promoter controls TPI expression did not grow at all on medium containing 1.6- 1.8 mM diamide. Moreover, yeast cells ectopically express- ing yeast TPI from the same promoter, which is approximately 30% more active than human wild-type TPI in yeast [11], were even more sensitive to diamide. Thus, diminishing the expression level or activity of TPI increases the diamide tolerance of yeast. Next, we investigated whether this phenomenon is con- served in multicellular eukaryotes, and addressed this by using Caenorhabditis elegans as a model. RNA interference (RNAi) technology was used to reduce (knock down) the intracellular concentration of TPI by feeding worms with E. coli producing double-stranded RNA of the C. elegans tpi-1 gene (Y17G7B.7); the empty RNAi vector (L4440) was used as control. The reduction of the intracellular TPI concentra- tion was analyzed by immunoblotting (Figure 1b, left panel). Then, tpi-1 knock-down worms were placed on agar plates supplemented with the oxidant juglone (5-hydroxy- 1,4-naphthalenedione, CAS No. 481-39-0), a natural naph- thoquinone found particularly in the black walnut Juglans nigra. This oxidant triggers the generation of superoxide rad- icals as a result of its capacity for redox cycling that involves a one-electron redox reaction generating semiquinone and superoxide radicals [21]. As controls, multi-stress-resistant http://jbiol.com/content/6/4/10 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. 10.3 Journal of Biology 2007, 6:10 Figure 1 Reduced triosephosphate isomerase (TPI) activity increases oxidant resistance of S. cerevisiae and C. elegans. (a) The left panel shows a Western blot analysis of yeast cells expressing wild-type human TPI under the control of promoters of different strengths: GPD1 (GPD pr ), TEF1 (TEF pr ), and CYC1 (CYC pr ). Yeast cells expressing human TPI Ile170Val or yeast TPI under the control of the strong GPD1 promoter were used as controls. Equal loading of the lysates was controlled by visualizing G6PDH. The right panel shows yeast cells expressing yeast TPI and human TPI Ile170Val controlled by the GPD1 promoter or yeast expressing wild-type human TPI controlled by the GPD1, TEF1 or CYC1 promoters, respectively. Yeast were spotted as fivefold serial dilutions on SC medium supplemented with different concentrations of diamide. Plates were incubated at 30°C for 3 days. (b) The left panel shows western blot analysis of cell extracts prepared from adult C. elegans that were fed with E. coli producing double-stranded RNA of the C. elegans tpi-1 gene (Y17G7B.7) (tpi-1 RNAi) or harboring the empty plasmid L4440 (control). The right panel shows the effects of the oxidants juglone and diamide on these worms. After feeding with E. coli as described above, worms were placed on agar plates supplemented with juglone or diamide. Multi-resistant daf-2 (e1370) mutant worms were included in every experiment as controls. 0 1 2 3 4 5 6 7 8 Animals alive (percentage) 0 10 20 30 40 50 60 70 80 90 100 Animals alive (percentage) 0 10 20 30 40 50 60 70 Wild-type tpi-1 RNAi daf-2 (e1370) 80 90 100 012 Without diamide 1.4 mM diamide 1.6 mM diamide 1.8 mM diamide 345 DiamideJuglone 6789 GPD pr -TPI lle170Val GPD pr -TPI lle170Val GPD pr -yeast TPI GPD pr -yeast TPI GPD pr -TPI GPD pr -TPI TEF pr -TPI Control tpi-1 RNAi TEF pr -TPI CYC pr -TPI CYC pr -TPI TPI TPI-1 DAF-21 G6PDH (a) (b) Time (h) Time (h) daf-2 mutant worms were included in every experiment, and surviving worms were counted each hour. Worms with reduced TPI concentration placed on 10 µM juglone plates survived significantly longer than wild-type animals under the same conditions (Figure 1b, middle panel). In addition, the average survival time of wild-type worms on 10 µM juglone plates was 4.2 ± 0.8 hours, whereas TPI knock- down animals survived for 5.5 ± 0.4 hours (p-value of 1.13e -07 , see Additional data file 1 for more quantitative information). We also carried out the same set of experi- ments using the oxidant diamide, which is not usually used in C. elegans laboratories. We discovered that worms were highly resistant to this oxidant, and very high concentra- tions had to be applied for growth inhibition (data not shown). Notably, we showed, by applying as much as 250 mM diamide, that TPI knock-down worms displayed an increased resistance (Figure 1b, right panel). The knock-down of TPI resulted in a greater average survival time compared with wild-type animals (8.6 ± 0.3 hours vs 7.5 ± 0.3 hours, p-value of 0.011, see Additional data file 1). Thus, these experiments clearly show that a reduction in TPI activity increases oxidant resistance of the multicellular eukaryote C. elegans. Reduced TPI activity protects against diamide by increasing the activity of the PPP We next aimed to dissect the molecular basis for the observed diamide resistance in yeast by genetic means. The glycolytic pathway is directly interconnected with the PPP, which is one of the key pathways in reducing the pyridine nucleotide NADP + to NADPH within the eukaryotic cyto- plasm and, hence, one of the main cellular sources of the cytoplasmic NADPH that is required as a redox cofactor by the main antioxidant enzymes to neutralize ROS (see [18] for a review). We speculated that the inactivation of TPI, resulting in a block on glycolysis, should counteract oxida- tive stress by elevating the metabolic flux of the PPP (Figure 2a). To test this assumption, we aimed to genetically target the first two steps of the PPP. As indicated in Figure 2a, the rate-limiting generation of D-6-phospho- glucono-δ-lactone from glucose-6-phosphate (g6p), the metabolite for which glycolysis and PPP are competing for, is catalyzed by the yeast glucose-6-phosphate dehydroge- nase (G6PDH) Zwf1p [17,22]. In the second step of the PPP, this metabolite is converted by the paralogous 6- phospho-gluconolactonases Sol3p and Sol4p into 6-phos- phogluconate [23]. Blocking these two essential steps would impair the activity of the PPP and lessen the observed pro- tective effect of reduced TPI activity. We therefore generated yeast strains expressing wild-type human TPI or TPI Ile170Val in which the yeast genes TPI1 and ZWF1, TPI1 and SOL3, or TPI1 and SOL4 were deleted. These strains were then spotted as fivefold serial dilutions on synthetic media containing different concentrations of diamide. As shown in Figure 2b, growth of the correspond- ing ∆tpi1∆zwf1, ∆tpi1∆sol3 and ∆tpi1∆sol4 yeast cells was strongly reduced compared with the respective ∆tpi1 yeast cells on medium containing 1.4-2.0 mM diamide. Notably, ∆tpi1∆zwf1 cells, which are unable to metabolize g6p to enter the PPP, exhibited the strongest sensitivity; these cells already grew poorly on medium supplemented with 1.2 mM diamide. As expected, ∆tpi1 yeast cells expressing TPI Ile170Val grew better on media containing high diamide concentrations compared with yeast cells expressing wild- type TPI, confirming the protective effect observed earlier. Strikingly, the protective effect of TPI Ile170Val was no longer observed in ∆tpi1∆zwf1 cells, in which the interplay between glycolysis and the PPP is blocked. In addition, the protective effect of TPI Ile170Val against diamide in ∆tpi1∆sol3 and ∆tpi1∆sol4 cells was detectable, but weaker. This was expected, since ∆tpi1∆sol3 and ∆tpi1∆sol4 cells are still able to convert D-6-phospho-glucono-δ-lactone to 6-phosphogluconate by reducing one equivalent of NADP + due to the presence of one wild-type copy of either SOL4 or SOL3. Thus, these experiments clearly demonstrate that the protective effect of reduced TPI activity is indeed based on the activity of the PPP and is absent if the first and rate-limiting step of the PPP is inhibited. Preventing the accumulation of NADPH sensitizes yeast cells to diamide As most antioxidant enzymes are coupled to NADPH as a redox cofactor and a functional defense mechanism against oxidative stress depends upon the availability of NADPH, we hypothesized that increased activity of the PPP might protect against oxidative stress due to the enhanced cellular produc- tion of this molecule. To test this hypothesis, we set out to measure the overall NADPH/NADP + ratio of MR101 cells expressing human wild-type TPI and MR105 cells expressing TPI Ile170Val . The respective strains were grown in duplicate to mid-log phase and pyridine nucleotides were extracted simul- taneously as described by Noack et al. [24], performing a three-step protocol that is based on a 34:24:1 phenol:chloro- form:isoamyl-alcohol pyridine-nucleotide extraction that is followed by two diethylether re-extractions of the aqueous phase. As measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS), the overall NADPH/NADP + ratio was indeed highly increased in MR105 cells expressing TPI Ile170Val in comparison to MR101 cells expressing wild-type TPI (Figure 3a). Although the LC-MS/MS analysis does not allow discrimination between cytoplasmic and mitochond- rial NADP(H), the measurements clearly show that the redox equilibrium of the NADP(H) pool strongly shifts towards NADPH in cells with reduced TPI activity; the increase in the sole cytoplasmic NADPH/NADP + ratio is expected to be 10.4 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. http://jbiol.com/content/6/4/10 Journal of Biology 2007, 6:10 even higher than the measured values of the overall NADPH/NADP + ratio. To substantiate these results in vivo and to correlate with the observed oxidant-resistance phenotype, we investigated the effect of the Gdp1 protein of the yeast Kluyveromyces lactis, a phosphorylating (NADP + -dependent) glyceraldehyde-3- phosphate dehydrogenase (GenBank accession number CAD23142, Enzyme Commission classification EC 1.2.1.13 [25]). Except for K. lactis, this enzyme has not been detected in non-plant eukaryotes; it was discovered in a screen designed to find suppressors for the lethal effects of phos- phoglucose isomerase (Pgi1) deletion in S. cerevisiae on glucose media [25]. The absence of Pgi1p is lethal for S. cerevisiae on standard media, because a strong NADPH accu- mulation occurs at the expense of its oxidized form [26]. http://jbiol.com/content/6/4/10 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. 10.5 Journal of Biology 2007, 6:10 Figure 2 Reduced TPI activity protects against diamide by increasing the metabolic flux through the PPP. (a) Schematic illustration of a subset of biochemical reactions of the glycolytic pathway (left) and the associated pentose phosphate pathway (right). Solid lines represent direct, one-step biochemical reactions, and indirect, multi-step reactions are represented as dotted lines. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (b) Yeast deletion strains ∆tpi1∆zwf1, ∆tpi1∆sol3, and ∆tpi1∆sol4 expressing wild-type human TPI or TPI Ile170Val were spotted as fivefold serial dilutions on synthetic media supplemented with different concentrations of diamide, and plates were incubated at 30°C. Glucose NADP + NADP + NAD + AT P ADP NADPH H + NADPH H + NADH H + Glycolysis Pentose phosphate pathway Erythrose-4-phosphate ZWF1 TPI SOL3 SOL4 Glucose-6- phosphate Glycerol-3- phosphate Fructose-1,6- bisphosphate 6-Phospho- gluconate Ribulose-5- phosphate Dihydroxyacetone- phosphate Glyceraldehyde-3- phosphate 1,3-Bisphospho-glycerate Citrate cycle Sedoheptulose-7- phosphate D-6-phospho- glucono-δ-lactone Xylulose-5- phosphate Ribose-5- phosphate NADP + NADPH H + ∆tpi1 ∆tpi1 ∆tpi1 ∆sol3 ∆sol4 ∆tpi1 ∆zwf1 TPI lle170Val TPI TPI lle170Val TPI TPI lle170Val TPI TPI lle170Val TPI Without diamide 1.2 mM diamide 1.4 mM diamide 1.8 mM diamide 2.0 mM diamide (a) (b) GAPDH Expression of K. lactis Gdp1 rescued the lethality of ∆pgi1 S. cerevisiae cells because it catalyzes the oxidation of NADPH to NADP + [25], thus preventing the accumulation of NADPH in ∆pgi1 cells [25]. Gdp1 can therefore be applied in vivo to analyze the impact of NADPH accumulation in regard to the observed oxidant resistance of yeast cells with reduced TPI activity. To do this, we transformed the yeast strain BY4741 with either a plasmid encoding K. lactis GDP1 under the control of a constitutive promoter or with an empty control plasmid and selected the respective transfor- mants on plates of synthetic complete (SC) medium lacking uracil (SC -ura ). Yeast cultures were then grown and spotted 10.6 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. http://jbiol.com/content/6/4/10 Journal of Biology 2007, 6:10 Figure 3 Reduced TPI activity protects against diamide by increasing NADPH. (a) S. cerevisiae strains MR101 and MR105 were grown in duplicate to mid-log phase, pyridine nucleotides were extracted, and LC-MS/MS measurements were performed in triplicate. MR105 cells expressing TPI Ile170Val had a higher overall NADPH/NADP + ratio compared with MR101 cells expressing wild-type TPI. (b) S. cerevisiae strain BY4741 was transformed with an empty 2µ plasmid or with a 2µ plasmid encoding K. lactis GDP1 (p1696). Afterwards, single transformants were selected, grown overnight and the same number of cells were spotted as fivefold serial dilutions on agar plates supplemented with different concentrations of diamide. Growth was monitored after plates were incubated at 30°C for 3 days. (c) The isogenic yeast strains MR101 expressing wild-type human TPI or MR105 expressing human TPI Ile170Val were transformed with plasmids for expression of K. lactis GDP1 and processed as described in (b). Vector TPI TPI TPI + Gdp1 TPI lle170Val TPI lle170Val + Gdp1 TPI TPI + Gdp1 TPI lle170Val TPI lle170Val + Gdp1 TPI TPI + Gdp1 TPI lle170Val TPI lle170Val + Gdp1 Without diamide Without diamide 1.6 mM diamide 2.0 mM diamide 2.2 mM diamide 1.8 mM diamide 2.0 mM diamide 0.0 0.1 0.2 Overall NADPH/NADP + ratio 0.3 TPI lle170Val Gdp1 Vector Gdp1 Vector Gdp1 Vector Gdp1 (a) (c) (b) as fivefold dilution series on solid medium supplemented with varying concentrations of diamide. As shown in Figure 3b, yeast cells expressing Gdp1 were highly sensitive to diamide in the concentration range of 1.8-2.0 mM com- pared with control cells, indicating that the cellular NADPH/NADP + balance is crucial for the cellular resistance to diamide. To further validate that increased activity of the PPP leading to an elevated cellular reduction of NADP + to NADPH underlies the observed resistance to diamide, we addressed the impact of GDP1 expression in ∆tpi1 yeast strains expressing the human protein TPI Ile170Val . We observed that growth of ∆tpi1 yeast expressing the human TPI proteins and K. lactis Gdp1 was strongly impaired on medium supplemented with 2.0 or 2.2 mM diamide (Figure 3c). Remarkably, the effects of GDP1 expression were less dramatic in yeast cells expressing TPI Ile170Val , which have an increased NADPH/NADP + ratio. Thus, these results suggest that the enhanced diamide resistance of yeast cells with reduced TPI activity is based on increased conversion of NADP + to NADPH within the PPP. Inactivation of TPI and GAPDH increases the concentration of PPP metabolites We observed in an earlier study [11] that yeast cells with reduced TPI activity are not resistant to oxidative stress caused by hydroperoxides such as hydrogen peroxide (H 2 O 2 ), cumene hydroperoxide or tert-butylhydroperoxide. Strikingly, treatment of yeast cells with these oxidants leads to a rapid inactivation of GAPDH; however, this inactivation is not observed when cells are treated with diamide [6,27]. As GAPDH is the first enzyme downstream of TPI, we specu- lated that the block of GAPDH activity in hydroperoxide- treated yeast cells prevents the protective effects of reduced TPI activity. This hypothesis would imply that cells do inac- tivate GAPDH to reroute the metabolic flux to the PPP for protection against ROS. To corroborate this, we comprehen- sively measured a number of glycolytic and PPP metabo- lites, and compared changes between their intracellular concentration in yeast cells expressing TPI variants with reduced activity and wild-type yeast cells treated with H 2 O 2 . For this analysis, the corresponding yeast cultures were grown in rich medium (YPD) to an equal optical density and lysed as described in Materials and methods. In the quantitative metabolomic analyses, we focused on the metabolites dhap, glucose-6-phosphate/fructose-6-phos- phate (g6p), 6-phosphogluconate (6pg), ribose-5-phosphate (r5p), xylulose-5-phosphate/ribulose-5-phosphate (x5p), sedoheptulose-7-phosphate (s7p), glyceraldehyde-3-phos- phate (gly3p) and glycerol-3-phosphate (gol3p). Quantifi- cation was carried out using LC-MS/MS. We first set out to analyze the experimental quality of our measurements, and prepared two samples from each culture for measurements of the various metabolites. The measure- ments of the parallel samples were plotted on a two-dimen- sional graph and analyzed statistically (Figure 4a, upper panel). The coefficient of determination (R²) equaled 0.9989 when including all measurements (0.98 for values smaller than 10), indicating high reproducibility of the analysis. Next, we assayed the comparability of the metabo- lite content of yeast cultures cultivated in duplicate. Two lysate samples of each culture were prepared in parallel and the metabolite content of each sample was measured in duplicate. The average concentration of each metabolite was plotted on a two-dimensional graph and analyzed statisti- cally (Figure 4a, lower panel). Here, the R² value of 0.995 (0.96 analyzing values smaller than 10) demonstrated excel- lent comparability of the metabolite content of yeast cul- tures grown in parallel. Finally, we calculated the relative alterations in the cellular metabolite concentrations of two different yeast strains - MR101, which expresses human TPI, and MR105, which expresses human TPI Ile170Val - compared with the isogenic wild-type strain BY4741 (Figure 4b, upper panel). MR101 yeast exhibits 70% and MR105 20% overall TPI activity compared with the wild-type strain BY4741, as determined by the TPI activity assay described earlier [11]. As expected, we detected increased levels of the TPI substrate dhap in yeast cells with reduced TPI activity, as previously observed in human cell extracts and in yeast [13,14]. The moderately reduced TPI activity in MR101 cells caused an increase in the intracellular dhap concentration of 24.9% compared with the level of wild-type strain BY4741. A strong increase in dhap concentration was measured in lysates prepared from MR105 cells and we also found that the concentration of g6p was increased in MR101 and MR105 cells. As men- tioned previously, g6p is converted by glycolysis and the PPP and is rate-limiting for their activity (Figure 2a). In addition, the intracellular concentration of the metabolites 6pg, r5p and x5p, all generated in the PPP, were elevated in MR101 and MR105 cells. Notably, the concentration changes of these metabolites followed the trend in TPI activ- ity in both these strains: the lower the TPI activity, the higher the increase in metabolite concentration. As expected, the cellular concentration of the TPI product, gly3p, was decreased in both strains. Furthermore, the metabolite s7p was decreased in MR101 cells, but increased in MR105 cells. This unexpected finding could potentially reflect a change in the equilibrium between gly3p and s7p, as both metabolites are simultaneously required by the yeast transketolases Tkl1p and Tkl2p; however, an adequate explanation cannot be given at present. Thus, these experi- ments clearly show that a decrease in cellular TPI activity results in elevated levels of almost all the metabolites of the PPP. http://jbiol.com/content/6/4/10 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. 10.7 Journal of Biology 2007, 6:10 10.8 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. http://jbiol.com/content/6/4/10 Journal of Biology 2007, 6:10 Figure 4 TPI and GAPDH inactivation increases the concentration of PPP metabolites. (a) For quality control of the metabolite quantifications and for analyzing the technical reproducibility, each metabolite was measured in duplicate (top panel). For analyzing the biological reproducibility, the metabolite concentrations were measured from cultures grown in parallel (bottom panel). Please note that for the purpose of illustration values greater than 10 are not shown. The complete plots are presented in Additional data file 3. (b) Upper panel, changes in metabolite levels in yeast strains with differing TPI activity. Lysates of yeast strains BY4741 (100% TPI activity), MR101 (70% TPI activity) and MR105 (20% TPI activity) were prepared and metabolites were quantified by LC-MS/MS. The absolute metabolite concentrations of MR101 and MR105 yeast were normalized and plotted as change given in percent relative to the wild-type (BY4741) strain. Middle panel, changes in metabolite levels in yeast with GAPDH inactivation. Cultures of strain BY4741 were treated with H 2 O 2 or left untreated. The relative changes of the various metabolites of the H 2 O 2 -treated cells in comparison to untreated cells were plotted. Bottom panel, predicted qualitative changes in metabolite concentrations using the non-fitted metabolic model. Note that for technical reasons, the abbreviation g6p refers to the sum of glucose-6-phosphate and fructose- 6-phosphate and x5p to the sum of xylulose-5-phosphate and ribulose-5-phosphate. (c) Upper panel, GAPDH activity in yeast cells treated with and without H 2 O 2 as in (b). Lower panel, effect of H 2 O 2 on wild-type yeast cells transformed with the 2µ plasmids p423GPD, p423GPD-EcoGAP encoding E. coli GAPDH, or p423GPD-TDH3 encoding the yeast GAPDH Tdh3p. Transformants were selected, grown overnight and the same number of cells were spotted as fivefold serial dilutions on SC -his-ade media supplemented with H 2 O 2 as indicated. R 2 = 0.98 R 2 = 0.96 8 6 4 2 0 0246 810 +250% Reduced TPI activity Inactivated GAPDH Metabolic modeling Relative change compared with wild-type +200% +150% +100% dhap g6p 6pg r5p x5p s7p gly3p gol3p dhap dhap g6p g6p 6pg 6pg 509.9 700.3 2435.8 r5p r5p x5p x5p s7p s7p gly3p gly3p gol3p +50% 0% −50% +250% 100% 0 Relative change compared with wild-type +200% +150% +100% + 0 − +50% 0% −50% 10 8 6 4 2 0 0246 Culture B Normal Vector EcoGAP TDH3 Vector EcoGAP TDH3 GAPDH activity Measurement B Measurement ACulture A 810 MR101 (70% TPI activity) MR105 (20% TPI activity) Reduced TPI activity Reduced GAPDH activity H 2 O 2 H 2 O 2 Without H 2 O 2 0.2 mM H 2 O 2 (a) (c) (b) 10 We next analyzed whether treatment of yeast cells with H 2 O 2 , known to cause inactivation of GAPDH [10,27,28], would result in a similar rerouting of the carbohydrate flux. Wild-type cells were treated with H 2 O 2 for 30 minutes as described [28], collected by centrifugation, and the GAPDH activity was measured as described in Materials and methods. As shown in Figure 4c (upper panel), GAPDH was inactivated in H 2 O 2 -treated yeast cells. To further demon- strate the contribution of GAPDH to resistance to H 2 O 2 , we investigated the H 2 O 2 -tolerance of yeast cells overexpressing either the most abundant yeast GAPDH paralog, Tdh3p, or the E. coli GAPDH, EcoGAP. As anticipated, cells over- expressing Tdh3p or EcoGAP were more sensitive to H 2 O 2 treatment compared with cells harboring the empty vector (Figure 4c, lower panel). Moreover, Tdh3p or EcoGAP over- expression in another yeast background, the W303 derivate Y2546, also caused sensitivity to H 2 O 2 (data not shown). Subsequently, we analyzed the changes in metabolite con- centrations of H 2 O 2 -treated yeast cells and found that con- centrations of all measured PPP metabolites were greatly increased (Figure 4b, middle panel). The greatest increases were observed for 6pg, x5p and s7p. Moreover, we found decreased concentrations of the glycolytic metabolite gol3p, which is generated intracellularly from dhap by the enzyme Gpd1p (also known as Hor1p). Strikingly, all measured metabolites showed a similar tendency in the case of inacti- vated GADPH as was observed for low TPI activity, with the exception of gly3p. Indeed, gly3p represents the metabolic intermediate of both enzymes. These results show that yeast cells reroute the carbohydrate flux in response to H 2 O 2 treatment in the same manner as cells with low TPI activity. This implies that rerouting of the metabolic flux is a basic mechanism in counteracting oxidative stress that is natu- rally switched on in the course of GAPDH inactivation. Mathematical modeling and computer simulations Because our experimental data imply that inactivation of GAPDH may serve as a cellular switch to reroute the meta- bolic flux from glycolysis to the PPP under oxidative stress conditions, we set out to develop a mathematical model that describes the dynamic behavior of the metabolic reac- tions under consideration. Most of the reactions involved have been intensely studied in vitro and, hence, sufficient kinetic parameters (K m , V max ) are available for modeling and simulating the entire pathway in silico. For this, we modeled enzymatic reactions (see Additional data file 2) as a set of ordinary differential equations using the CellDe- signer software [29]. The model allows calculation of the concentrations of 19 different metabolites, the amount of high-energy phosphate groups (P), and the NAD + /NADH and NADP + /NADPH ratios. Three types of in silico simula- tions were run: with normal TPI and GAPDH activity; with 25% residual TPI activity; and with 25% residual GAPDH activity. The results of these simulations were compared with the LC-MS/MS measurements from wild-type yeast, from strain MR105, which expresses TPI Ile170Val , and from H 2 O 2 -treated wild-type cells with inactivated GAPDH. The simulations revealed that 13 of the 14 qualitative changes in metabolite concentrations were correctly predicted by the mathematical model (Figure 4b, lower panel). A difference between the experimental data and the predictions was only observed for the metabolite s7p. The simulations predicted a decline of s7p in H 2 O 2 -treated yeast cells whereas the respective experiments showed that the concentration of s7p increased. As the qualitative predictions of the unfitted model matched well with the experimental data set, we calculated the influence of reduced TPI or GAPDH activity on the cel- lular NADPH/NADP + ratio without any further parameter fitting. Like other mathematical models [30,31], our model is based on the fact that the nicotinamide nucleotide moiety is conserved: that is, that the sum of cellular NADP + and NADPH is constant. The free-energy change (∆G) of a reac- tion is given by ∆G = ∆G 0’ +RT·ln(k), (where ∆G 0’ is the stan- dard free-energy change and k is the equilibrium constant). For a redox reaction involving NADPH and NADP + (k = reduced form/oxidized form), it is therefore the NADPH/NADP + ratio, and not the absolute concentrations, that drives the reaction. Hence, we calculated the corre- sponding steady-state values of the NADPH/NADP + ratio depending on the activity of GAPDH or TPI using the program Copasi 4B20 [32] (Figure 5a). Reduction of TPI activity resulted in an increased NADPH/NADP + ratio from approximately 6.5 to 9. The simulated reduction in GAPDH activity resulted in an even greater increase in the NADPH/NADP + ratio, from approximately 6.5 to 19. Taken together, simulations using a dynamic, unfitted mathemati- cal model corroborate the experimental finding that reduced TPI and GAPDH activities redirect the carbohydrate flux. Moreover, the model predicts an elevated NADPH/NADP + ratio if the activity of the PPP is increased, a result that agrees with earlier experimental observations. Although the qualitative results of the simulations fit very well with the measurements without modifying any of the kinetic parameters taken from the literature, it should be noted that the kinetic constants were determined using enzymes from five different species (human, cow, rabbit, yeast, E. coli) in different laboratories over a period of more than three decades. Consequently, it cannot be expected that the simulations coincide quantitatively with the mea- sured metabolite concentrations. However, the high-quality LC-MS/MS data allowed us to adjust the numerical values of the kinetic parameters so that the predicted metabolite con- centrations agree better with the measured ones. For this http://jbiol.com/content/6/4/10 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. 10.9 Journal of Biology 2007, 6:10 10.10 Journal of Biology 2007, Volume 6, Article 10 Krobitsch et al. http://jbiol.com/content/6/4/10 Journal of Biology 2007, 6:10 Figure 5 In silico model for the interplay of glycolysis and the pentose phosphate pathway in response to GAPDH or TPI inactivation. (a) Predicted changes of the cytoplasmic NADPH/NADP + ratio of the unfitted model. The NADPH/NADP + ratio increases in correlation with the rate of TPI (blue) or GAPDH (red) inactivation. (b) Quantitative accuracy of the metabolic model before and after parameter fitting. Upper panel, 21 measured metabolite concentrations (seven metabolites under three conditions) are plotted against the predicted values before fitting. Lower panel, data versus prediction after parameter fitting. (c) As (a), but after parameter fitting. (d) Comparison of quantitative predictions made with the parameter-fitted and the measured metabolite concentrations. Changes relative to the wild-type strain values are shown. Black and red bars correspond to yeast cells with inactivated GAPDH, green and yellow bars correspond to reduced TPI activity. Before fitting Predicted concentrations (mM) Predicted concentrations (mM) Measured concentrations (mM) NADPH/NADP + (GAPDH) NADPH/NADP + (TPI) NADPH/NADP + (GAPDH) NADPH/NADP + (TPI) 25 30 35 40 45 50 55 10.0 9.0 8.5 8.0 7.5 7.0 6.5 6.0 26 28 30 32 34 60 0 20 40 60 80 0.0 0.5 1.0 1.5 2.0 Measured concentrations (mM) 0.0 0.5 1.0 1.5 2.0 100 After fitting Before fitting After fitting Enzyme inactivation (percentage) 0 20 40 60 80 100 Enzyme inactivation (percentage) 0 2 0 0.5 1.0 1.5 2.0 4 6 8 Percentage change to wild type 4 6 8 10 12 14 16 18 20 22 dhap g6p 800 600 400 200 0 6pg r5p x5p s7p gly3p GAPDH data GAPDH model TPI data TPI model (a) (c) (d) (b) [...]... the discovery that a reduction in intracellular TPI activity results in enhanced oxidant resistance in S cerevisiae and C elegans, we directly addressed the question of whether blockage of TPI causes a redirection of the metabolic flux from glycolysis to the PPP By genetic means, we showed that the oxidant resistant phenotype of cells with reduced TPI activity is based on the activity of the PPP; this... as well as to a large number of genetic and infectious diseases Therefore, understanding the mechanisms that counteract the cellular consequences of oxidative stress is of immense interest, in particular in the perspective that enhancing cellular tolerance of eukaryotic cells to oxidative stress may result in the identification of proteins exploitable as therapeutic targets In this light, the glucose... rerouting of the carbohydrate flux represents an immediate key to counteracting oxidative stress Although earlier studies reported that an enhanced activity of the well-conserved PPP, which is strongly interconnected with the glycolytic pathway, was observed in mammalian cells under conditions of oxidative stress [7,15], the underlying cellular mechanism is far from being understood Encouraged by the. .. redirecting the carbohydrate flux Volume 6, Article 10 Krobitsch et al 10.11 from glycolysis to the PPP Notably, our modeling approach revealed that the experimentally observed alteration in s7p levels cannot be explained by the current knowledge of the kinetics of glycolysis and PPP It would be therefore of interest to focus in future on the sedoheptulose metabolism in order to close this gap The good... electrochemical potential of the cell, is responsible for the enhanced oxidant tolerance Because ROS provoke a shift of the cellular redox state, which is often defined as the balance of the overall NADH/NAD+ and NADPH/NADP+ ratios, a central task in counteracting oxidative damage is to maintain the cytoplasmic NADPH/NADP+ ratio For this process, enzymes of the PPP are crucial In contrast to NAD(H), whose redox equivalents... reroutes the carbohydrate flux to maintain the cytoplasmic NADPH/NADP+ equilibrium to counteract oxidative stress In addition, it is fairly likely that the altered levels of metabolites act as an early signaling event in cell-cycle progression and control, as it has been shown that GAPDH activity is a main regulator of H2O2-induced apoptosis [41] In general, oxidative stress contributes profoundly to the. .. agreement between the model and the experimental results underlines the solidness of the model and provides a firm base for further comprehensive simulations of eukaryotic carbohydrate metabolism integrating other metabolic pathways that are associated with glycolysis and the PPP PPP activity is a regulator of normal lifespan of S cerevisiae and C elegans Much evidence exists that oxidative damage to diverse... effect is absent in yeast cells in which the first and rate-limiting step of the PPP is inhibited In addition, our metabolic datasets clearly support the idea that decreasing the cellular TPI activity leads to raised levels of PPP metabolites We also provide experimental and in silico evidence that increased reduction of NADP+ to NADPH within the PPP, which raises the electrochemical potential of the. .. available with this article online Additional data file 1 contains details of the C elegans experiment Additional data file 2 contains a figure and a table of the reactions included in the mathematical model to study the effects of a diminished TPI or GAPDH activity on the Journal of Biology 2007, 6:10 http://jbiol.com/content/6/4/10 Journal of Biology 2007, flux through glycolysis and the pentose phosphate... between mitochondria and cytoplasm [38], the cellular pools of NADP(H) seem to be maintained independently; NADPH generated in the cytoplasm is not available to mitochondria, and vice versa In addition, cytoplasmic and mitochondrial NADP(H) are synthesized independently from NAD(H) by different NAD and NADH kinases [39] Moreover, the intracellular concentration of NADP(H) is low compared with that of NAD(H); . activity of the PPP might protect against oxidative stress due to the enhanced cellular produc- tion of this molecule. To test this hypothesis, we set out to measure the overall NADPH/NADP + ratio of. PPP, which is one of the key pathways in reducing the pyridine nucleotide NADP + to NADPH within the eukaryotic cyto- plasm and, hence, one of the main cellular sources of the cytoplasmic NADPH that is. elegans and that the underlying mechanism is based on a redirection of the metabolic flux from glycolysis to the pentose phosphate pathway, altering the redox equilibrium of the cytoplasmic NADP(H)